Process for manipulating the level of glycan content of a glycoprotein

ABSTRACT

The present invention provides a method for manipulating the fucosylated glycan content on a recombinant protein.

BACKGROUND OF INVENTION

A variety of post-translational modifications including methylation,sulfation, phosphorylation, lipid addition and glycosylation areperformed on proteins expressed by higher eukaryotes. Glycosylationinvolves the covalent attachment of sugar moieties to specific aminoacids and is one of the most common and important posttranslationalmodification for recombinant proteins. Protein glycosylation plays arole in multiple functions, including protein folding and qualitycontrol, molecular trafficking and sorting, and cell surface receptorinteraction. Many of the secreted proteins, membrane proteins andproteins targeted to vesicles or certain intracellular organelles areknown to be glycosylated.

While glycosylation can take many forms, N-linked glycosylation is themost common. N-linked glycosylation involves addition ofoligosaccharides to an asparagine residue found in certain recognitionsequences in proteins (e.g., Asn-X-Ser/Thr). N-linked glycoproteinscontain standard branched structures which are composed of mannose,galactose, N-acetylglucosamine and neuramic acids. N-linkedglycosylation of the Fc domain of recombinantly expressed therapeuticantibodies is a critical posttranslational modification. Typicaltherapeutic antibodies have complex glycoforms possessing fucosylatedbi-antennary glycans with a trimannosyl core capped by anN-acetylgalactosamine (GlcNAc), galactose, and N-acetylneuraminic acid(Neu5Ac) residue on each branch. Other glycoforms may be afucosylated,galactosylated, sialylated, have terminal or bisecting GlcNAc, have highmannose (5-9 residues), etc.

Glycosylation can affect therapeutic efficacy of recombinant proteindrugs. It is well known that variations in Fc glycosylation can affectFc-mediated effector functions. Some glycoforms, such as galactosylationand sialylation, are desirable for decreasing immunogenicity, andothers, such as afucosylation, bisecting GlcNAc residues, and highmannose glycans, enhance antibody-dependent cellular cytotoxicity (ADCC)activity.

Glycosylation is important in the determination of the structure andfunction of therapeutic antibodies. It determines binding capabilitiesand often determines the recognition and processing of the antibody onceit is introduced in a therapeutic application. In the case ofgalactosylation and fucosylation, they determine the complementdependent cytotoxicity (CDC) activity and ADCC functions, respectively,that they influence.

The level of β-galactosylation is related to more “mature” glycoforms.Galactose addition is one of the last stages of glycosylation that takesplace in the Golgi apparatus before secretion. Terminal galactose isneeded for sialylation, which may be the final step in the glycosylationof some proteins. Galactose also serves as a ligand for galactosebinding proteins and is the basis of a variety of antigenic responseswhich are related to carbohydrate content. Galactose has also been shownto impact the conformation of the protein in solution. (Furukawa andSato, (1999) Biochimica et Biophysica Acta (BBA), 1473 (1), pages 54-86and Houde et al., (2010) Molecular and Cellular Proteomics, 9(8), pages1716-1728.

Fucosylation also takes place in the Golgi apparatus as part of thematuration of the protein prior to secretion. If a protein isfucosylated it typically happens before galactosylation in theglycosylation pathway. However, fucosylation is not necessary forgalactosylation to proceed (Hossler et al., (2009)_Glycobiology, 19(9),pages 936-949).

The influence of glycosylation on bioactivity, pharmacokinetics,immunogenicity, solubility and in vivo clearance of therapeuticglycoproteins have made monitoring and control of glycosylation acritical parameter for biopharmaceutical manufacturing. Therefore,methods for manipulating the level of glycan content of therapeuticproteins would be beneficial.

There is a need in the pharmaceutical industry to manipulate and controlthe level of glycan content of recombinant therapeutic glycoproteins andmethods for accomplishing such without significant impact on cellgrowth, viability and productivity would be useful. The inventionprovides a method for manipulating the fucosylated glycan content on arecombinant protein by regulating copper and manganese content and pH incell culture medium.

SUMMARY OF THE INVENTION

The invention provides a method for manipulating the fucosylated glycancontent on a recombinant protein comprising inoculating a bioreactorwith host cells expressing the recombinant protein, culturing the hostcells in a serum free, chemically defined cell culture medium; whereinthe cell culture medium includes from 10 to 100 ppb copper and from 50to 1000 nM manganese, at pH 7.0, harvesting the recombinant proteinproduced by the host cell, wherein the level of afucosylated glycans onthe recombinant protein increases compared to the afucosylated glycanlevel obtained in the same cell culture medium at a lower pH.

In one embodiment the method further comprising an increase in the levelof β-galactosylation on the recombinant protein.

In one embodiment the concentration of coper is 100 ppb.

In one embodiment the concentration of manganese is 1000 nM.

In one embodiment the fucosylated glycan content is manipulated toinfluence the effector function of the recombinant protein.

In one embodiment the method further comprising a temperature shift. Ina related embodiment the temperature shift is from 36° C. to 31° C. Inanother related embodiment the temperature shift occurs at thetransition between the growth phase and production phase. In yet anotherrelated embodiment the temperature shift occurs during the productionphase.

In one embodiment the host cell expressing the recombinant protein iscultured in a batch culture, fed-batch culture, perfusion culture, orcombinations thereof. In a related embodiment the culture is a perfusionculture. In another related embodiment perfusion comprises continuousperfusion. In another related embodiment rate of perfusion is constant.In another related embodiment the perfusion is performed at a rate ofless than or equal to 1.0 working volumes per day. In yet anotherrelated the perfusion is accomplished by alternating tangential flow.

In one embodiment the bioreactor has a capacity of at least 500 L.

In one embodiment the bioreactor has a capacity of at least 500 L to2000 L.

In one embodiment the bioreactor has a capacity of at least 1000 L to2000 L.

In one embodiment the bioreactor is inoculated with at least 0.5 × 10⁶cells/mL.

In one embodiment the serum-free chemically defined cell culture mediumis a perfusion cell culture medium.

In one embodiment the host cells are mammalian cells.

In one embodiment the host cells are Chinese Hamster Ovary (CHO) cells.

In one embodiment the recombinant protein is a glycoprotein.

In one embodiment the recombinant protein is selected from the groupconsisting of a human antibody, a humanized antibody, a chimericantibody, a recombinant fusion protein, or a cytokine.

In one embodiment the recombinant protein produced by the host cell ispurified and formulated into a pharmaceutically acceptable formulation.

In one embodiment is a recombinant protein produced by the method of theinvention. In a related embodiment the recombinant protein according ispurified. In yet another related embodiment the recombinant protein isformulated into a pharmaceutically acceptable formulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Integrated Viable Cell Density (10⁶ cell days/ml)

-   pH 6.85 50 Mn²⁺ 10 Cu²⁺ (gray dashed line with +)-   pH 6.85 50 Mn²⁺ 100 Cu²⁺ (gray line with +)-   pH 6.85 1000 Mn²⁺ 10 Cu²⁺ (gray dashed line with open circle)-   pH 6.85 50 Mn²⁺ 100 Cu²⁺ (gray line with open circle)-   pH 7.0 50 Mn²⁺ 10 Cu²⁺ (black dashed line with +)-   pH 7.0 50 Mn²⁺ 100 Cu²⁺ (black line with +)-   pH 7.0 1000 Mn²⁺ 10 Cu²⁺ (black dashed line with open circle)-   pH 7.0 50 Mn²⁺ 100 Cu²⁺ (black line with open circle)-   pH appeared to be the only factor impacting cell growth.    Concentrations of manganese and copper seemed to have no effect on    cell growth.

FIG. 2 Viability (%)

-   pH 6.85 50 Mn²⁺ 10 Cu²⁺ (gray dashed line with +)-   pH 6.85 50 Mn²⁺ 100 Cu²⁺ (gray line with +)-   pH 6.85 1000 Mn²⁺ 10 Cu²⁺ (gray dashed line with open circle)-   pH 6.85 50 Mn²⁺ 100 Cu²⁺ (gray line with open circle)-   pH 7.0 50 Mn²⁺ 10 Cu²⁺ (black dashed line with +)-   pH 7.0 50 Mn²⁺ 100 Cu²⁺ (black line with +)-   pH 7.0 1000 Mn²⁺ 10 Cu²⁺ (black dashed line with open circle)-   pH 7.0 50 Mn²⁺ 100 Cu²⁺ (black line with open circle)

By the day 17 the cultures at pH 6.85 had higher final viabilitycompared to cultures run at pH 7.0. However, final viability was over80%, regardless of pH. Copper and manganese concentration in the rangestested had no effect on viability.

FIG. 3 Packed cell adjusted titer (g/L)

-   pH 6.85 50 Mn²⁺ 10 Cu²⁺ (gray dashed line with +)-   pH 6.85 50 Mn²⁺ 100 Cu²⁺ (gray line with +)-   pH 6.85 1000 Mn²⁺ 10 Cu²⁺ (gray dashed line with open circle)-   pH 6.85 50 Mn²⁺ 100 Cu²⁺ (gray line with open circle)-   pH 7.0 50 Mn²⁺ 10 Cu²⁺ (black dashed line with +)-   pH 7.0 50 Mn²⁺ 100 Cu²⁺ (black line with +)-   pH 7.0 1000 Mn²⁺ 10 Cu²⁺ (black dashed line with open circle)-   pH 7.0 50 Mn²⁺ 100 Cu²⁺ (black line with open circle)-   pH appears to have no statistical impact on packed cell adjusted    titer, likewise, copper and manganese concentration had no effect on    this cell line and process.

FIG. 4 Viable Cell Density (10⁵ cell days/ml)

-   pH 6.85 50 Mn 10 Cu (gray dashed line with +)-   pH 6.85 50 Mn 100 Cu (gray line with +)-   pH 6.85 1000 Mn 10 Cu (gray dashed line with open circle)-   pH 6.85 50 Mn²⁺ 100 Cu²⁺ (gray line with open circle)-   pH 7.0 50 Mn²⁺ 10 Cu²⁺ (black dashed line with +)-   pH 7.0 50 Mn²⁺ 100 Cu²⁺ (black line with +)-   pH 7.0 1000 Mn²⁺ 10 Cu²⁺ (black dashed line with open circle)-   pH 7.0 50 Mn²⁺ 100 Cu²⁺ (black line with open circle)

Reactors run at pH 6.85 grew to cell densities of nearly 10⁶ cells permL more than reactors grown at pH 7.0. Concentration of copper andmanganese had no statistically significant effect on cell growth in thisexperiment for this cell line and process. pH was the only factorimpacting cell growth.

FIG. 5 β-Galactosylation (Adj. R² = 0.95) Prediction profiler generatedusing JMP statistical software. The profile illustrates thedirectionality and magnitude of the changes in β-galactosylation as aresult of manipulating pH and manganese concentration. The terms in theprofiler represent the remaining terms in the statistical model afterremoving those terms that were not statistically significant. Theaddition of manganese had a significant effect on the level ofbeta-galactosylation; the greater the concentration of manganese, thegreater the percentage of beta-galactosylation. pH also had astatistically significant effect on beta-galactosylation, as pHincreased, beta-galactosylation also increased, but not to the extentobserved when manganese was added.

FIG. 6 Afucosylation (Adj. R² = 0.92) Prediction profiler generatedusing JMP statistical software. The profile illustrates thedirectionality and magnitude of the changes in afucosylation as a resultof manipulating pH, manganese and copper concentrations. The terms inthe profiler represent the remaining terms in the statistical modelafter removing those terms that were not statistically significant.Copper, manganese and pH all had a statistically significant impact onthe afucosylation levels. Increasing levels of copper and manganese, aswell as increasing pH, all resulted in an increase in afucosylation.

FIG. 7 ADCC relative cytotoxicity, at base afucosylation (4%), 6%afucosylation and 8% afucosylation.

FIG. 8 CDC scaled dose response, at base β-galactosylation, (2.7%), 25%β-galactosylation and 50% β-galactosylation.

DETAILED DESCRIPTION OF THE INVENTION

Varying the concentration of manganese in cell culture medium caninfluence the degree of β-galactosylation of recombinant antibodies.Manganese acts as a cofactor in the modulation of the activity ofgalactosyltransferase. The galactosyltransferase mediated reactionemploys UDP-galactose as the sugar substrate and manganese as thecofactor. A change in the level of galactosylation can be caused by achange in the UDP-galactose availability or a change in the enzymaticactivity (for example, by altering the manganese cofactorconcentration), or both.

Analogously, fucosylation may be moderated by altering the levels of theGDP-fucose substrate, by interfering with the activity offucosyltransferase or by modifying the GDP-fucose transporter mechanism.However, metal ions have not been reported to play a direct role in anyof these mechanisms. As described herein, increasing the level ofmanganese and copper was found to impact recombinant protein fucosylatedglycan content by significantly increasing the level of afucosylatedglycans. In addition it was found that pH also played a major role indetermining glycosylation patterns.

The type and extent of N-linked glycosylation on IgG1 antibodies areknown to affect Fc-mediated effector functions. For example, the levelof afucosylation strongly enhances antibody dependent cell mediatedcytotoxicity (ADCC) by increasing binding affinity to Fcy receptors,whereas the level of galactosylation can influence complement dependentcytotoxicity (CDC) activity. This makes it critical to understand andcontrol the nature and level of glycosylation of therapeutic proteins.As described herein, the enhancement of afucosylation andgalactosylation had substantial impact on ADCC and CDC effector

A method is provided to improve control of the levels of afucosylatedglycans on a recombinant protein by manipulating the pH and theconcentrations of manganese (Mn²⁺) and copper (Cu²⁺) in a cell culturemedium. The levels of afucosylated and β-galactosylated glycans wereincreased without impacting cell culture performance.

The invention provides a method for manipulating the fucosylated glycancontent on a recombinant protein comprising inoculating a bioreactorwith host cells expressing the recombinant protein, culturing the hostcells in a serum free, chemically defined cell culture medium; whereinthe cell culture medium includes from 10 to 100 ppb copper and from 50to 1000 nM manganese, at pH 7.0, harvesting the recombinant proteinproduced by the host cell, wherein the level of afucosylated glycans onthe recombinant protein increases compared to the afucosylated glycanlevel obtained in the same cell culture medium at a lower pH. In oneembodiment the method further comprising an increase in the level ofβ-galactosylation on the recombinant protein. In one embodiment theconcentration of copper is 100 ppb. In one embodiment the concentrationof manganese is 1000 nM. In one embodiment the fucosylated glycancontent is manipulated to influence the effector function of therecombinant protein.

In one embodiment the method further comprising a temperature shift. Ina related embodiment the temperature shift is from 36° C. to 31° C. Inanother related embodiment the temperature shift occurs at thetransition between the growth phase and production phase. In yet anotherrelated embodiment the temperature shift occurs during the productionphase.

In one embodiment the host cell expressing the recombinant protein iscultured in a batch culture, fed-batch culture, perfusion culture, orcombinations thereof. In a related embodiment the culture is a perfusionculture. In another related embodiment perfusion comprises continuousperfusion. In another related embodiment rate of perfusion is constant.In another related embodiment the perfusion is performed at a rate ofless than or equal to 1.0 working volumes per day. In yet anotherrelated the perfusion is accomplished by alternating tangential flow.

In one embodiment the bioreactor has a capacity of at least 500 L. Inone embodiment the bioreactor has a capacity of at least 500 L to 2000L. In one embodiment the bioreactor has a capacity of at least 1000 L to2000 L. In one embodiment the bioreactor is inoculated with at least 0.5× 10⁶ cells/mL.

In one embodiment the serum-free chemically defined cell culture mediumis a perfusion cell culture medium. In one embodiment the host cells aremammalian cells. In one embodiment the host cells are Chinese HamsterOvary (CHO) cells.

In one embodiment the recombinant protein is a glycoprotein. In oneembodiment the recombinant protein is selected from the group consistingof a human antibody, a humanized antibody, a chimeric antibody, arecombinant fusion protein, or a cytokine. In one embodiment therecombinant protein produced by the host cell is purified and formulatedinto a pharmaceutically acceptable formulation. In one embodiment is arecombinant protein produced by the method of the invention. In arelated embodiment the recombinant protein according is purified. In yetanother related embodiment the recombinant protein is formulated into apharmaceutically acceptable formulation.

Cell Culture

By “cell culture” or “culture” is meant the growth and propagation ofcells outside of a multicellular organism or tissue. Suitable cultureconditions for mammalian cells are known in the art. See e.g. Animalcell culture: A Practical Approach, D. Rickwood, ed., Oxford UniversityPress, New York (1992). Mammalian cells may be cultured in suspension orwhile attached to a solid substrate.

As used herein, the terms “cell culturing medium” (also called “culturemedium”, “cell culture media”, “tissue culture media”) refers to anynutrient solution used for growing cells, e.g., animal or mammaliancells, and which generally provides at least one or more components fromthe following: an energy source (usually in the form of a carbohydratesuch as glucose); one or more of all essential amino acids, andgenerally the twenty basic amino acids, plus cysteine; vitamins and/orother organic compounds typically required at low concentrations; lipidsor free fatty acids; and trace elements, e.g., inorganic compounds ornaturally occurring elements that are typically required at very lowconcentrations, usually in the micromolar range.

The nutrient solution may optionally be supplemented with additionalcomponents to optimize growth of cells, such as hormones and othergrowth factors, e.g., insulin, transferrin, epidermal growth factor,serum, and the like; salts, e.g., calcium, magnesium and phosphate, andbuffers, e.g., HEPES; nucleosides and bases, e.g., adenosine, thymidine,hypoxanthine; and protein and tissue hydrolysates, e.g., hydrolyzedanimal protein (peptone or peptone mixtures, which can be obtained fromanimal byproducts, purified gelatin or plant material); antibiotics,e.g., gentamycin; cell protectants or surfactants, polyamines, e.g.,putrescine, spermidine or spermine (see e.g., WIPO Publication No. WO2008/154014) and pyruvate (see e.g. U.S. Pat. No. 8053238) depending onthe requirements of the cells to be cultured and/or the desired cellculture parameters.

Non-ionic surfactants may also be added to the cell culture medium.Examples of non-ionic surfactants include, but are not limited to,polyvinyl alcohol, polyethylene glycol, and non-ionic block copolymersurfactants. Also included are alkyl poly(ethylene oxide), copolymers ofpoly(ethylene oxide) and poly(propylene oxide) (EO-PO block copolymers),poly(vinylpyrrolidone), alkyl polyglucosides (such as sucrosemonostearate, lauryl diglucoside, or sorbitan monolaureate, octylglucoside and decyl maltoside), fatty alcohols (cetyl alcohol or olelylalcohol), or cocamides (cocamide MEA, cocamide DEA and cocamide TEA).

Also included are block copolymers based on ethylene oxide and propyleneoxide, also referred to as polyoxypropylene-polyoxyethylene blockcopolymers. These molecules are nonionic triblock copolymers having acentral hydrophobic chain of polyoxypropylene (poly(propylene oxide))flanked by two hydrophilic chains of polyoxyethylene (poly(ethyleneoxide)). Of particular interest are those having 70 polyoxypropyleneunits and 30 units of each of the polyoxyethylene chains. In a preferredembodiment the block copolymer is poloxamer 188 (CAS #90003-11-6 with anaverage molecular weight of 8.4 kd, BASF Chemical, Washington, NJ) whichis sold under various brand names such as Pluronic® F68, Kolliphor®P-188, Lutrol® F68, and Lutrol® 188. Such non-ionic surfactants may beadded at concentrations up to 5 g/L or more and may be used to maintaincell viability for longer culture durations under ATF perfusionconditions.

The present invention provides a cell culture medium that contains from10 to 100 ppb copper and from 50 to 1000 nM manganese. In one embodimentthe cell culture medium contains 100 ppb manganese. In anotherembodiment the cell culture medium contains 1000 nM manganese. Inanother embodiment the cell culture medium contains 100 ppb manganeseand 1000 nM manganese. Copper and manganese salts useful for thisinvention include, but are not limited to, cupric sulfate pentahydrateand manganese sulfate monohydrate.

Cell culture medium components, including copper and manganese, may becompletely milled into a powder medium formulation; partially milledwith liquid supplements added to the cell culture medium as needed; orcell culture medium components may be added in a completely liquid formto the cell culture.

Cell culture medium include those that are typically employed in and/orare known for use with any cell culture process, such as, but notlimited to, batch, extended batch, fed-batch and/or perfusion orcontinuous culturing of cells.

A “base” (or batch) cell culture medium refers to a cell culture mediumthat is typically used to initiate a cell culture and is sufficientlycomplete to support the cell culture.

A “growth” cell culture medium refers to a cell culture medium that istypically used in cell cultures during a period of exponential growth, a“growth phase”, and is sufficiently complete to support the cell cultureduring this phase. A growth cell culture medium may also containselection agents that confer resistance or survival to selectablemarkers incorporated into the host cell line. Such selection agentsinclude, but are not limited to, geneticin (G4118), neomycin, hygromycinB, puromycin, zeocin, methionine sulfoximine, methotrexate,glutamine-free cell culture medium, cell culture medium lacking glycine,hypoxanthine and thymidine, or thymidine alone.

A “production” cell culture medium refers to a cell culture medium thatis typically used in cell cultures during the transition and productionphases when exponential growth is ending and protein production takesover, and is sufficiently complete to maintain a desired cell density,viability and/or product titer during these phases.

A “perfusion” cell culture medium refers to a cell culture medium thatis typically used in cell cultures that are maintained by perfusion orcontinuous culture methods and is sufficiently complete to support thecell culture during this process. Perfusion cell culture mediumformulations may be enriched or more concentrated than base cell culturemedium formulations to accommodate for the method used to remove thespent medium. Perfusion cell culture medium can be used during both thegrowth and production phases.

Cell cultures can be supplemented with concentrated feed mediumcontaining components, such as nutrients and amino acids, which areconsumed during the course of the production phase of the culture.Concentrated cell culture medium can contain some or all of thenutrients necessary to maintain the cell culture; in particular,concentrated medium can contain nutrients identified as or known to beconsumed during the course of the production phase of the cell culture.Concentrated medium may be based on just about any cell culture mediaformulation. Such a concentrated feed medium can contain some or all thecomponents of the cell culture medium at, for example, about 2X, 3X, 4X,5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 25X, 30X, 40X, 50X, 75X,100x, 200X, 400X, 600X, 800X, or even 1000X of their normal amount.

Cell culture medium, in certain embodiments, may be serum-free and/orfree of products or ingredients of animal origin. Cell culture medium,in certain embodiments, may be chemically defined, where all of thechemical components are known.

As is appreciated by the practitioner, animal or mammalian cells arecultured in a medium suitable for the particular cells being culturedand which can be determined by the person of skill in the art withoutundue experimentation. Commercially available media can be utilized andinclude, but is not limited to, Iscove’s Modified Dulbecco’s Medium,RPMI 1640, and Minimal Essential Medium-alpha. (MEM-alpha), Dulbecco’sModification of Eagle’s Medium (DMEM), DME/F12, alpha MEM, Basal MediumEagle with Earle’s BSS , DMEM high Glucose, with Glutamine, DMEM highglucose, without Glutamine, DMEM low Glucose, without Glutamine,DMEM:F12 1:1, with Glutamine, GMEM (Glasgow’s MEM), GMEM with glutamine,Grace’s Complete Insect Medium, Grace’s Insect Medium, without FBS,Ham’s F-10, with Glutamine, Ham’s F-12, with Glutamine, IMDM with HEPESand Glutamine, IMDM with HEPES and without Glutamine, IP41 InsectMedium, 15 (Leibovitz)(2X), without Glutamine or Phenol Red, 15(Leibovitz), without Glutamine, McCoy’s 5A Modified Medium, Medium 199,MEM Eagle, without Glutamine or Phenol Red (2X), MEM Eagle-Earle’s BSS,with glutamine, MEM Eagle-Earle’s BSS, without Glutamine, MEMEagle-Hanks BSS, without Glutamine, NCTC-109, with Glutamine, Richter’sCM Medium, with Glutamine, RPMI 1640 with HEPES, Glutamine and/orPenicillin-Streptomycin, RPMI 1640, with Glutamine, RPMI 1640, withoutGlutamine, Schneider’s Insect Medium or any other media known to oneskilled in the art, which are formulated for particular cell types. Tothe foregoing exemplary media can be added supplementary components oringredients, including optional components, in appropriateconcentrations or amounts, as necessary or desired, and as would beknown and practiced by those having in the art using routine skill.

Cell cultures can also be supplemented with independent concentratedfeeds of particular nutrients which may be difficult to formulate or arequickly depleted in cell cultures. Such nutrients may be amino acidssuch as tyrosine, cysteine and/or cystine (see e.g., WIPO PublicationNo. 2012/145682). For example, a concentrated solution of tyrosine maybe independently fed to a cell culture grown in a cell culture mediumcontaining tyrosine. A concentrated solution of tyrosine and cystine mayalso be independently fed to the cell culture being grown in a cellculture medium lacking tyrosine, cystine and/or cysteine. Theindependent feeds can begin prior to or at the start of the productionphase. The independent feeds can be accomplished by fed batch to thecell culture medium on the same or different days as the concentratedfeed medium. The independent feeds can also be perfused on the same ordifferent days as the perfused medium.

Methods can be employed to continuous feed a mammalian cell culture,such as those that do not employ feedback control (see WIPO PublicationNo. WO 2013/040444).

Media Treatments

The cell culture medium can be treated using methods or devices tosterilize or disinfect media prior to addition to the bioreactor and/orcell culture. Cell culture media may be treated using high temperatureshort time (HTST) (see, e.g., U.S. Pat. No. 7,420,183). Cell culturemedia may also be treated using UV in combination with filtration (see,e.g., WIPO Publications WO 2008/157247; WO 2012/115874; WO 2013/063298and WO 2013/138159). Cell culture media may be subjected tonanofiltration (see, e.g., Liu et al., (2000) Biotechnol. Prog.16:425-434). Cell culture media may be treated with chemicals thatinactivate viruses, such as solvents, detergents, psoralen, orbeta-propiolactone.

Cells

Cell lines (also referred to as “host cells”) used in the invention aregenetically engineered to express a polypeptide of commercial orscientific interest. Cell lines are typically derived from a lineagearising from a primary culture that can be maintained in culture for anunlimited time. The cells can contain introduced, e.g., viatransformation, transfection, infection, or injection, expressionvectors (constructs), such as plasmids and the like, that harbor codingsequences, or portions thereof, encoding the proteins for expression andproduction in the culturing process. Such expression vectors contain thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to and practiced by thoseskilled in the art can be used to construct expression vectorscontaining sequences encoding the produced proteins and polypeptides, aswell as the appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in J. Sambrook et al., 2012, Molecular Cloning, ALaboratory Manual, 4^(th) edition Cold Spring Harbor Press, Plainview,N.Y. or any of the previous editions; F. M. Ausubel et al., 2013,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y, or any of the previous editions; Kaufman, R.J., Large ScaleMammalian Cell Culture, 1990, all of which are incorporated herein forany purpose.

Animal cells, mammalian cells, cultured cells, animal or mammalian hostcells, host cells, recombinant cells, recombinant host cells, and thelike, are all terms for the cells that can be cultured according to theprocesses of this invention. Such cells are typically cell linesobtained or derived from mammals and are able to grow and survive whenplaced in either monolayer culture or suspension culture in mediumcontaining appropriate nutrients and/or other factors, such as thosedescribed herein. The cells are typically selected that can express andsecrete proteins, or that can be molecularly engineered to express andsecrete, large quantities of a particular protein, more particularly, aglycoprotein of interest, into the culture medium. It will be understoodthat the protein produced by a host cell can be endogenous or homologousto the host cell. Alternatively, the protein is heterologous, i.e.,foreign, to the host cell, for example, a human protein produced andsecreted by a Chinese hamster ovary (CHO) host cell. Additionally,mammalian proteins, i.e., those originally obtained or derived from amammalian organism, are attained by the methods the present inventionand can be secreted by the cells into the culture medium.

The method of the present invention can be used in the culture of avariety of cells. In one embodiment, the cultured cells are eukaryoticcells such as plant and/or animal cells. The cells can be mammaliancells, fish cells, insect cells, amphibian cells or avian cells. A widevariety of mammalian cell lines suitable for growth in culture areavailable from the American Type Culture Collection (Manassas, Va.) andother depositories as well as commercial vendors. Cell that can be usedin the processes of the invention include, but not limited to, MK2.7cells, PER-C6 cells, Chinese hamster ovary cells (CHO), such as CHO-K1(ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet.,12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and WO01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/-DHFR,Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), anddp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney cells (CV1, ATCCCCL-70); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7,ATCC CRL-1651); HEK 293 cells, and Sp2/0 cells, 5L8 hybridoma cells,Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkatcells, THP-1 cells, Sp2/0 cells, primary epithelial cells (e.g.,keratinocytes, cervical epithelial cells, bronchial epithelial cells,tracheal epithelial cells, kidney epithelial cells and retinalepithelial cells) and established cell lines and their strains (e.g.,human embryonic kidney cells (e.g., 293 cells, or 293 cells subclonedfor growth in suspension culture, Graham et al., 1977, J. Gen. Virol.,36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse sertolicells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervicalcarcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCCCCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells(HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51);buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather,1982, Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells; PER-C6retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells,BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells,WISH cells, BS-C-I cells, LLC-MK₂ cells, Clone M-3 cells, 1-10 cells,RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁ cells, PK(15) cells, GH₁cells, GH₃ cells, L2 cells, LLC-RC 256 cells, MH₁C₁ cells, XC cells,MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof),fibroblast cells from any tissue or organ (including but not limited toheart, liver, kidney, colon, intestines, esophagus, stomach, neuraltissue (brain, spinal cord), lung, vascular tissue (artery, vein,capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow,and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g.,TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells,Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells,Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells,WI-38 cells, WI-26 cells, MiCli cells, CV-1 cells, COS-1 cells, COS-3cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCCCRL-1587; VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB/3T3 cells, F9cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells,NOR-10 cells, C₃H/IOTI/2 cells, HSDM₁C₃ cells, KLN205 cells, McCoycells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L)cells, L-MTK (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells,Swiss/3T3 cells, Indian muntac cells, SIRC cells, C_(II) cells, andJensen cells, or derivatives thereof)or any other cell type known to oneskilled in the art.

Cells may be suitable for adherent, monolayer or suspension culture,transfection, and expression of proteins, for example, antibodies. Thecells can be used with batch, fed batch and perfusion or continuousculture methods.

Types of Cell Cultures

For the purposes of understanding, yet without limitation, it will beappreciated by the skilled practitioner that cell cultures and culturingruns for protein production can include three general types; namely,batch or extended batch culture, fed-batch culture, perfusion culture,or combinations thereof. In batch culture, cells are initially culturedin medium and this medium is not removed, replaced, or supplemented,i.e., the cells are not “fed” with fresh medium, during or before theend of the culturing run. The desired product is harvested at the end ofthe culturing run.

For fed-batch cultures, the culturing run time is increased bysupplementing the culture medium one or more times (or continuously)with fresh medium during the run, i.e., the cells are “fed” with newmedium (“fed medium”) during the culturing period. Fed-batch culturescan include the various feeding regimens and times, for example, daily,every other day, every two days, etc., more than once per day, or lessthan once per day, and so on. Further, fed-batch cultures can be fedcontinuously with feeding medium. The desired product may then beharvested at the end of the culturing/production run.

Perfusion culture, sometimes referred to as continuous culture, is onein which the cell culture receives fresh perfusion medium, and wherespent medium is removed from the bioreactor during the run. Perfusion offresh media into the cell culture and removal of spend media can becontinuous, step-wise, intermittent, or a combination of any or all ofthese. Perfusion rates can range from less than one working volume perday to many working volumes per day.

The term “perfusion flow rate” is the amount of media that is passedthrough (added and removed) from a bioreactor, typically expressed assome portion of or a multiple of the working volume, in a given time.The perfusion flow rate may vary over the duration of the cell culturerun. “Working volume” refers to the amount of bioreactor volume used forcell culture. In one embodiment the perfusion flow rate is less than orequal to one working volume per day. Perfusion feed medium can beformulated to maximize perfusion nutrient concentration to minimizeperfusion rate.

Preferably the cells are retained in the culture and the spent mediumthat is removed is substantially free of cells or has significantlyfewer cells than the culture. Recombinant proteins expressed by the cellculture can also be retained in the culture for later harvest or removedwith the spent medium.

Perfusion can be accomplished by a number of means includingcentrifugation, sedimentation, or filtration, See e.g. Voisard et al.,(2003), Biotechnology and Bioengineering 82:751-65. In one embodiment afiltration method is used. Filters include membrane filters, ceramicfilters and metal filters and may be in any shape, including spiralwound or tubular or in the form of a sheet. One or more filters can beconnected to, in fluid communication with, a bioreactor together orindependently, in series or in parallel.

Hollow fiber filters may be used in mammalian cell perfusion culture forcell and/or recombinant protein retention. When the cell culture,including cell culture media, cells (whole and lysed), soluble expressedrecombinant proteins, host cell proteins, waste products and the like,are introduced to the filter, depending on the pore size or molecularweight cutoff (MWCO) the hollow fiber material may retain certain cellculture components on the lumen side (inside) and allow certaincomponents to pass through the filter (permeate) based on the pore sizeor molecular weight cutoff of the hollow fiber material. The materialthat is retained (retentate) is returned to the bioreactor. Freshperfusion cell culture media is added to the bioreactor and permeate iswithdrawn from the filter at predetermined intervals or continuously tomaintain a desired or constant bioreactor volume. The permeate can bediscarded, stored in holding tanks, bags or totes or transferreddirectly to another unit operation, such as filtration, flocculation,centrifugation and/or other downstream purification methods or the like.Hollow fibers for microfiltration typically have a pore size rangingfrom 0.1 µm to 5-10 µm or a molecular weight cut off of 500 kDa or moreand can be used to allow the protein to pass through into the permeate.Ultrafiltration hollow fibers typically have a pore size range of 0.01µm to 0.1 µm or a molecular weight cut off of 300 kDa or less, and canbe used to retain the desired protein in the retentate and return itback to the bioreactor. This can be used, for example, to concentratethe recombinant protein product for harvest. Such filters are availablecommercially, such as Xampler UFP-750-E-4MA, Xampler UFP-30-E-4MA, (GEHealthcare, Pittsburg, PA) and Midikros TC Modules T02-E030-10,T02-050-10, T02-E750-05, T02-M10U-06 (Spectrum Laboratories, Inc,Dominguez, CA).

The cell culture may be drawn out of the bioreactor and into the filterby a pumping system, which passes the cell culture through the lumenside of the hollow fiber. Examples of cell pumping systems includeperistaltic pumps, double diaphragm pumps, low shear pumps (Levitronix®pumps, Zurich, Switzerland) and alternating tangential flow systems(ATF™, Refine Technology, Pine Brook, NJ, See e.g. U.S. Pat. No.6,544,424; Furey (2002) Gen. Eng. News. 22 (7), 62-63). The permeate maybe drawn from the filters by use of peristaltic pumps. In a preferredembodiment perfusion is accomplished by use of an alternating tangentialflow system.

Cell Culture Processes

Cell culture can be carried out under conditions for small to largescale production of recombinant proteins using culture vessels and/orculture apparatuses that are conventionally employed for animal ormammalian cell culture. As is appreciated by those having skill in theart, tissue culture dishes, T-flasks and spinner flasks are typicallyused on a laboratory bench scale. For culturing on a larger scaleequipment such as, but not limited to, fermentor type tank culturedevices, air lift type culture devices, fluidized bed bioreactors,hollow fiber bioreactors, roller bottle cultures, stirred tankbioreactor systems, packed bed type culture devices, and single usedisposable bags or any other suitable devise known to one skilled in theart, can be used. Microcarriers may be used with the roller bottle orstirred tank bioreactor systems. The systems can be operated in a batch,fed-batch or perfusion/continuous mode. In addition, the cultureapparatus or system may be equipped with additional apparatus, such acell separators using filters, gravity, centrifugal force, and the like.

The production of recombinant proteins can be done in multiple phaseculture processes. In a multiple phase process, cells are cultured intwo or more distinct phases. For example cells may be cultured first inone or more growth phases, under environmental conditions that maximizecell proliferation and viability, then transitioned to a productionphase, under conditions that maximize protein production. In acommercial process for production of recombinant proteins by mammaliancells, there are commonly multiple, for example, at least about 2, 3, 4,5, 6, 7, 8, 9, 10 or more growth phases that occur in different culturevessels (N-x to N-1) preceding a final production culture. The growthand production phases may be preceded by, or separated by, one or moretransition phases. A production phase can be conducted at large scale.

The term “growth phase” of a cell culture refers to the period ofexponential cell growth (i.e., the log phase) where the cells aregenerally rapidly dividing. Cells are maintained at the growth phase fora period of about one day, or about two days, or about three days, orabout four days, or longer than four days. The duration of time forwhich the cells are maintained at growth phase will vary based on thecell-type and/or the rate of cell growth and/or the culture conditions,for example.

The term “transition phase” refers to a period of time between thegrowth phase and the production phase. Generally, transition phase isthe time during which culture conditions may be controlled to support ashift from growth phase to production phase. Various cell cultureparameters which may be controlled include but are not limited to, oneor more of, temperature, pH, osmolality, vitamins, amino acids, sugars,peptones, ammonium, salts and the like.

The term “production phase” of a cell culture refers to the period oftime where the cell growth has plateaued. The logarithmic cell growthtypically ends before or during this phase and protein production takesover. Fed batch and perfusion cell culture processes supplement the cellculture medium or provide fresh medium so as to achieve and maintaindesired cell density, viability and product titer at this stage. Aproduction phase can be conducted at large scale. Large scale cellcultures can be maintained in a volume of at least about 100, 500, 1000,2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters. In anembodiment of the invention, the production phase is conducted in 500 L,1000 L and/or 2000 L bioreactors.

Typically the cell cultures that precede a final production culture gothrough two prior phases, seed and inoculum trains. The seed train phase(N-X) takes place at small scale where cells are quickly expanded innumber. At the inoculums train phase (N-1), cells are further expandedto generate the inoculum for the production bioreactor, such as aninoculums of at least 0.5 × 10⁶ cells/mL. Seed and N-1 trains can beproduced by any culture method, typically batch cell cultures. N-1 celldensities of ≥0.5 × 10⁵ cells/mL are typical for seeding productionbioreactors. Higher N-1 cell densities can decrease or even eliminatethe time needed to reach a desired cell density in the productionbioreactor. A preferred method for achieving higher N-1 cell densitiesis perfusion culture using alternating tangential flow filtration. AnN-1 cell culture grown by means of a perfusion process using alternatingtangential flow filtration can provide cells at any desired density,such as densities of >90 × 10⁶ cells/mL or more. The N-1 cell culturecan be used to generate a single bolus inoculation cultures or can beused as a rolling seed stock culture that is maintained to inoculatemultiple production bioreactors. The inoculation density can have apositive impact on the level of recombinant protein produced. Productlevels tend to increase with increasing inoculation density. Improvementin titer is tied not only to higher inoculation density, but is likelyto be influenced by the metabolic and cell cycle state of the cells thatare placed into production. In one embodiment of the invention the cellculture is established by inoculating the bioreactor with at least 0.5 ×10⁶ cells/mL.

The term “cell density” refers to the number of cells in a given volumeof culture medium. “Viable cell density” refers to the number of livecells in a given volume of culture medium, as determined by standardviability assays (such as trypan blue dye exclusion method). The term“packed cell volume” (PCV), also referred to as “percent packed cellvolume” (%PCV), is the ratio of the volume occupied by the cells, to thetotal volume of cell culture, expressed as a percentage (see Stettler,et al., (2006) Biotechnol Bioeng. Dec 20:95(6): 1228-33). Packed cellvolume is a function of cell density and cell diameter; increases inpacked cell volume could arise from increases in either cell density orcell diameter or both. Packed cell volume is a measure of the solidlevel in the cell culture.

Cell Culture Controls

Cell culture conditions suitable for the methods of the presentinvention are those that are typically employed and known for batch,fed-batch, or perfusion (continuous) culturing of cells or anycombination of those methods, with attention paid to pH, dissolvedoxygen (O₂), and carbon dioxide (CO₂), agitation and humidity, andtemperature. During recombinant protein production it is desirable tohave a controlled system where cells are grown for a desired time or toa desired density and then the physiological state of the cells isswitched to a growth-limited or arrested, high productivity state wherethe cells use energy and substrates to produce the recombinant proteinin favor of increasing cell density. For commercial scale cell cultureand the manufacture of biological therapeutics, the ability to limit orarrest cell growth and being able to maintain the cells in agrowth-limited or arrested state during the production phase is verydesirable. Such methods include, for example, temperature shifts, use ofchemical inducers of protein production, nutrient limitation orstarvation and cell cycle inhibitors, either alone or in combination.

One such mechanism for limiting or arresting growth is to shift thetemperature during the cell culture. Temperature shifts may occur at anytime during the cell culture. A growth phase may occur at a highertemperature than a production phase. A cell culture may be run at afirst temperature set-point from about 35° C. to about 38° C., and thenthe temperature shifted to a second temperature set-point from about 29°C. to about 37° C., optionally from about 30° C. to about 36° C. or fromabout 30° C. to about 34° C. In one embodiment, a temperature shift mayoccur during the transition between the growth phase and the productionphase. In another embodiment, a temperature shift may occur during theproduction phase.

Switching the temperature set-point may be done manually or can be doneautomatically by making use of bioreactor control systems. Thetemperature set-point may be switched at a predetermined time or inresponse to one or more cell culture parameters, such as cell density,titer, or concentration of one or more media components. One such methoduses an online biomass monitoring tool integrated into the bioreactorcontrol system to trigger a temperature set-point change when a desiredcell density is reached. For example, a capacitance based biomass probemay be used for online cell density estimation and the data from onlinemeasurements can be used to trigger a shift in the bioreactortemperature. Such capacitance based probes include Fogale capacitancesensor (DN12-200) (Nimes, France).

Chemical inducers of protein production, such as caffeine, butyrate,and/or hexamethylene bisacetamide (HMBA), may be added independent of orat the same time as, before, or after a temperature shift. If inducersare added after a temperature shift, they can be added from one hour tofive days after the temperature shift, optionally from one to two daysafter the temperature shift. The cell cultures can then be maintainedfor days or even weeks while the cells produce the desired protein(s).

Another method to maintain cells at a desired physiological state is toinduce cell growth-arrest by exposure of the cell culture to lowL-asparagine conditions (see e.g., WIPO Publication No. WO2013/006479).Cell growth-arrest may be achieved and maintained through a culturemedium that contains a limiting concentration of L-asparagine andmaintaining a low concentration of L-asparagine in the cell culture.Maintaining the concentration of L-asparagine at 5 mM or less can beused to maintain cells in a growth-arrested state.

Cell cycle inhibitors, compound known or suspected to regulate cellcycle progression and the associated processes of transcription, DNArepair, differentiation, senescence and apoptosis related to this arealso useful to induce cell growth-arrest. Cell cycle inhibitors thatinteract with the cycle machinery, such as cyclin-dependent kinases(CDKs) are useful as are those molecules that interact with proteinsfrom other pathways, such as AKT, mTOR, and other pathways that affect,directly or indirectly, the cell cycle.

Harvest and Purification

The expressed recombinant proteins may be secreted into the culturemedium from which they can be recovered and/or collected. Therecombinant proteins may then be subjected to one or more processingsteps including harvest, purification, endotoxin and/or viralinactivation/filtration, and/or ultrafiltration/diafiltration.

The expressed recombinant proteins may be captured in the harvestpermeate. The proteins may be purified, or partially purified, fromharvest permeates using processes and commercially available productsknown in the art and/or available from commercial vendors. Such methodsinclude flocculation; centrifugation; precipitation; filtration methodssuch as depth filtration; chromatography methods including, affinitychromatography, size exclusion chromatography, ion exchangechromatography, mixed mode anion exchange chromatography, hydrophobicinteraction chromatography and hydroxyapatite chromatography, amongother available methods.

The purified proteins can then be “formulated”, meaning bufferexchanged, sterilized, bulk-packaged, and/or packaged for a final user.Suitable formulations for pharmaceutical compositions are known in theart and include those described in Remington’s Pharmaceutical Sciences,18th ed. 1995, Mack Publishing Company, Easton, PA.

Process Analytical Techniques

Process analytical technologies and methods are available to monitor andevaluate samples taken during cell culture and purification processes toquantitatively and/or qualitatively monitor characteristics of therecombinant protein and the production process. This real time or inlineinformation can be used to monitor and/or control product and productionparameters, such as titer, cell density; product quality attributes suchas post translational modifications; product or process variability suchas impurities and the like, to make timely decisions and modifyprocesses as necessary.

Each step of an upstream cell culture process or a downstreampurification process may be monitored to provide information about theamount of a particular product quality attribute (PQA) and to controlthis PQA with a preset target and range.

Samples may be taken intermittently, at desired frequencies, orcontinuously. Samples may be analyzed in real time or near real time orstored for later analysis. This information can be used to make changesduring upstream and downstream processes.

Detection of product quality attribute may be done using massspectrometry, liquid chromatography with UV and/or mass spectrometrydetection and capillary electrophoresis and the like.

These processes are adaptable to continuous monitoring with manual orautomated process adjustments such as feeds, temperature, processduration as determined by the level of a specified product qualityattribute.

Intact mass analysis to detect the presence of post-translationalmodifications such as amino acid processing and glycosylation may bemade using a polyhydroxyethyl aspartamide column operated insize-exclusion mode and coupled with ESI-MS (Brady et al., (2008) J AmSoc Mass Spectro, 19: 502-509)

Real-time monitoring eluate from ion exchange chromatography bymonitoring a normalized LS/UV ratio for each fraction using laser lightscattering detector and an UV absorbance, see U.S. Pat. Publication No.US 2013-0303732.

Multi-attribute method makes use of single liquid-chromatography/massspectrometry (LC/MS) to search and characterize tandem MS data usingvarious database and search platforms such as Sequest (The ScrippsResearch Institute, La Jolla, CA), X! Tandem (The Global ProteomeMachine Organization) or Mascot (Matrix Science, Boston, MA). Samplesmay be denatured at high pH or to maintain disulfide isoforms andprotect succinimide variants, at low pH. The sample is then reduced andalkylated followed by digestion with trypsin. The sample is theninjected into an MS (such as a Q Exactive™ Hybrid Quadrupole-OrbitrapMass Spectrometer, Thermo Fischer Scientific, Waltham, MA) and analysisis performed using Pinpoint software (Thermo Fischer Scientific).Attributes that can be identified, quantified and monitored includeisomerization, deamination, disulfide reduction, host cell proteincontamination, mutations, misincorporations, hydroxylysine, thioether,non-glycolysated heavy chains, C-terminal amidation, residual protein A,characterize glycans and provide molecule identity. The mass accuracyfor each attribute monitored can be set at less than 5 ppm of thepredicted mass. Identification of the peptide/attribute is confirmed byMS2 fragmentation and orthogonal characterization methods (HILIC-MS forglycosylation for example). The experimental isotopic distribution musthave a dot product score better than 0.95 when compared to thetheoretical isotopic distribution. A retention time window is set foreach attribute and all detectable charge states for each attribute areconsidered for quantification. A criteria is defined that will detectchanges in the attribute. For example, deamination can be monitored bydetermining a deamination value (deaminated peptide divided by the sumof the deaminated peptide and the unmodified parent peptide multipliedby 100. Glycosylation can be monitored by comparing each specific glycanto the sum of all detectable glycans.

Proteins

As used herein “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout and refer to a molecule comprising two ormore amino acid residues joined to each other by peptide bonds.Peptides, polypeptides and proteins are also inclusive of modificationsincluding, but not limited to, glycosylation resulting in glycoproteins,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation.

As used herein, the term “glycoprotein” refers to peptides and proteinshaving at least one oligosaccharide side chain including mannoseresidues. Glycoproteins may be homologous to the host cell, or may beheterologous, i.e., foreign, to the host cell being utilized, such as,for example, a human glycoprotein produced by a Chinese hamster ovary(CHO) host cell. Such glycoproteins are generally referred to as“recombinant glycoproteins.” In certain embodiments, glycoproteinsexpressed by a host cell are directly secreted into the medium.

Proteins can be of scientific or commercial interest, includingprotein-based drugs. Proteins include, among other things, antibodiesand fusion proteins. Peptides, polypeptides and proteins may be producedby recombinant animal cell lines using cell culture methods and may bereferred to as “recombinant peptide”, “recombinant polypeptide”,“recombinant protein”, “recombinant glycoprotein”. The expressedprotein(s) may be produced intracellularly or secreted into the culturemedium from which it can be recovered and/or collected.

Nonlimiting examples of mammalian proteins that can be advantageouslyproduced by the methods of this invention include proteins comprisingamino acid sequences identical to or substantially similar to all orpart of one of the following proteins: tumor necrosis factor (TNF), flt3ligand (WO 94/28391), erythropoeitin, thrombopoeitin, calcitonin, IL-2,angiopoietin-2 (Maisonpierre et al. (1997), Science 277(5322): 55-60),ligand for receptor activator of NF-kappa B (RANKL, WO 01/36637), tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, WO97/01633), thymic stroma-derived lymphopoietin, granulocyte colonystimulating factor, granulocyte-macrophage colony stimulating factor(GM-CSF, Australian Patent No. 588819), mast cell growth factor, stemcell growth factor (U.S. Pat. No.6,204,363), epidermal growth factor,keratinocyte growth factor, megakaryote growth and development factor,RANTES, human fibrinogen-like 2 protein (FGL2; NCBI accession no.NM_00682; Rüegg and Pytela (1995), Gene 160:257-62) growth hormone,insulin, insulinotropin, insulin-like growth factors, parathyroidhormone, interferons including α-interferons, γ-interferon, andconsensus interferons (U.S. Pat. Nos. 4,695,623 and 4,897471), nervegrowth factor, brain-derived neurotrophic factor, synaptotagmin-likeproteins (SLP 1-5), neurotrophin-3, glucagon, interleukins, colonystimulating factors, lymphotoxin-β, leukemia inhibitory factor, andoncostatin-M. Descriptions of proteins that can be produced according tothe inventive methods may be found in, for example, Human Cytokines:Handbook for Basic and Clinical Research, all volumes (Aggarwal andGutterman, eds. Blackwell Sciences, Cambridge, MA, 1998); GrowthFactors: A Practical Approach (McKay and Leigh, eds., Oxford UniversityPress Inc., New York, 1993); and The Cytokine Handbook, Vols. 1 and 2(Thompson and Lotze eds., Academic Press, San Diego, CA, 2003).

Additionally the methods of the invention would be useful to produceproteins comprising all or part of the amino acid sequence of a receptorfor any of the above-mentioned proteins, an antagonist to such areceptor or any of the above-mentioned proteins, and/or proteinssubstantially similar to such receptors or antagonists. These receptorsand antagonists include: both forms of tumor necrosis factor receptor(TNFR, referred to as p55 and p75, U.S. Pat. No. 5,395,760 and U.S. Pat.No. 5,610,279), Interleukin-1 (IL-1) receptors (types I and II; EPPatent No. 0460846, U.S. Pat. No. 4,968,607, and U.S. Pat. No.5,767,064,), IL-1 receptor antagonists (U.S. Pat. No. 6,337,072), IL-1antagonists or inhibitors (U.S. Pat. Nos. 5,981,713, 6,096,728, and5,075,222) IL-2 receptors, IL-4 receptors (EP Patent No. 0 367 566 andU.S. Pat. No. 5,856,296), IL-15 receptors, IL-17 receptors, IL-18receptors, Fc receptors, granulocyte-macrophage colony stimulatingfactor receptor, granulocyte colony stimulating factor receptor,receptors for oncostatin-M and leukemia inhibitory factor, receptoractivator of NF-kappa B (RANK, WO 01/36637 and U.S. Pat. No. 6,271,349),osteoprotegerin (U.S. Pat. No. 6,015,938), receptors for TRAIL(including TRAIL receptors 1, 2, 3, and 4), and receptors that comprisedeath domains, such as Fas or Apoptosis-Inducing Receptor (AIR).

Other proteins that can be produced using the invention include proteinscomprising all or part of the amino acid sequences of differentiationantigens (referred to as CD proteins) or their ligands or proteinssubstantially similar to either of these. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the VIth International Workshop andConference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD proteins are disclosed in subsequent workshops. Examples ofsuch antigens include CD22, CD27, CD30, CD39, CD40, and ligands thereto(CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are membersof the TNF receptor family, which also includes 41BB and OX40. Theligands are often members of the TNF family, as are 41BB ligand and OX40ligand.

Enzymatically active proteins or their ligands can also be producedusing the invention. Examples include proteins comprising all or part ofone of the following proteins or their ligands or a proteinsubstantially similar to one of these: a disintegrin andmetalloproteinase domain family members including TNF-alpha ConvertingEnzyme, various kinases, glucocerebrosidase, superoxide dismutase,tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E,apolipoprotein A-I, globins, an IL-2 antagonist, alpha-1 antitrypsin,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

The term “antibody” includes reference to both glycosylated andnon-glycosylated immunoglobulins of any isotype or subclass or to anantigen-binding region thereof that competes with the intact antibodyfor specific binding, unless otherwise specified, including human,humanized, chimeric, multi-specific, monoclonal, polyclonal, andoligomers or antigen binding fragments thereof. Also included areproteins having an antigen binding fragment or region such as Fab, Fab′,F(ab′)₂, Fv, diabodies, Fd, dAb, maxibodies, single chain antibodymolecules, complementarity determining region (CDR) fragments, scFv,diabodies, triabodies, tetrabodies and polypeptides that contain atleast a portion of an immunoglobulin that is sufficient to conferspecific antigen binding to a target polypeptide. The term “antibody” isinclusive of, but not limited to, those that are prepared, expressed,created or isolated by recombinant means, such as antibodies isolatedfrom a host cell transfected to express the antibody.

Examples of antibodies include, but are not limited to, those thatrecognize any one or a combination of proteins including, but notlimited to, the above-mentioned proteins and/or the following antigens:CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25,CD27L, CD32, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147,IL-1α, IL-1β, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-12, IL-12p35 subunit, IL-13, IL-21, IL-23, IL-23 p19 subunit, IL-12/IL-23 sharedp40 subunit, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13receptor, IL-17 receptor, IL-18 receptor subunits, FGL2, PDGF-β andanalogs thereof (see U.S. Pat. Nos. 5,272,064 and 5,149,792), B7RP-1,B7RP-2, VEGF, TGF, TGF-β2, TGF-β1, c-fms, EGF receptor (see U.S. Pat.No. 6,235,883), CGRP receptor, VEGF receptor, hepatocyte growth factor,proprotein convertase subtilisin/kexin type 9 (PCSK9), FGF21,osteoprotegerin ligand, interferon gamma, EGFRvIII, B lymphocytestimulator (BlyS, also known as BAFF, THANK, TALL-1, and zTNF4; see Doand Chen-Kiang (2002), Cytokine Growth Factor Rev. 13(1): 19-25), ST2,C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEMantigen, LCG (which is a gene product that is expressed in associationwith lung cancer), HER-2, HER-3, a tumor-associated glycoprotein TAG-72,the SK-1 antigen, tumor-associated epitopes that are present in elevatedlevels in the sera of patients with colon and/or pancreatic cancer,cancer-associated epitopes or proteins expressed on breast, colon,squamous cell, prostate, pancreatic, lung, and/or kidney cancer cellsand/or on melanoma, glioma, or neuroblastoma cells, the necrotic core ofa tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins,TSLP, IFNy, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α,the adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), angiopoietin 1 (Ang1),angiopoietin 2 (Ang2), leukointegrin adhesin, the platelet glycoproteingp IIb/IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2(which is an inhibitor of factor VIIa-tissue factor), MHC I,carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumor necrosisfactor (TNF), CTLA-4 (which is a cytotoxic T lymphocyte-associatedantigen), programmed cell death 1 (PD-1), programmed cell death ligand 1(PDL-1), programmed cell death ligand 2 (PDL-2), lymphocyte activationgene-3 (LAG-3), T-cell immunoglobulin domain and mucin domain 3 (TIM3),Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DR antigen, sclerostin, L-selectin,Respiratory Syncitial Virus, human immunodeficiency virus (HIV),hepatitis B virus (HBV), Streptococcus mutans, and Staphlycoccus aureus.Specific examples of known antibodies which can be produced using themethods of the invention include but are not limited to adalimumab,alirocumab, bevacizumab, infliximab, abciximab, alemtuzumab,bapineuzumab, basiliximab, belimumab, briakinumab, brodalumab,canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab,dupililumab, eculizumab, gemtuzumab guselkumab, ozogamicin, golimumab,ibritumomab, ixekizumab, ipilimumab, tiuxetan, labetuzumab,lebrikizumab, mapatumumab, mavrilimumab, matuzumab, mepolizumab,motavizumab, muromonab-CD3, nivolumab, natalizumab, nimotuzumab,ofatumumab, omalizumab, oregovomab, palivizumab, panitumumab,pemtumomab, pertuzumab, pembrolizumab, ranibizumab, rituximab,romosozumab, rovelizumab, rilotumumab, tildrakizumab, tocilizumab,tositumomab, tralokinumab, trastuzumab, tremelimumab, ustekinumab,vedolizomab, zalutumumab, and zanolimumab.

The invention can also be used to produce recombinant fusion proteinscomprising, for example, any of the above-mentioned proteins. Forexample, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Håkansson et al.(1999), Structure7:255-64. Specifically included among such recombinant fusion proteinsare proteins in which a portion of a receptor is fused to an Fc portionof an antibody such as etanercept (a p75 TNFR:Fc), and belatacept(CTLA4:Fc). Chimeric proteins and polypeptides, as well as fragments orportions, or mutants, variants, or analogs of any of the aforementionedproteins and polypeptides are also included among the suitable proteins,polypeptides and peptides that can be produced by the methods of thepresent invention. This includes trebananib, an angiopoietin (Ang) 1 and2 neutralizing peptibody. Also included are bi-specific T-cell engagers(BiTEs) that exert action selectively and direct the human immune systemto act against tumor cells. Specifically included among such BiTEs arethat target CD19, such as blinatumomab. Other molecules includeaflibercept.

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness to the meaning of the claims. Units, prefixes, andsymbols may be denoted in their SI accepted form. Numeric ranges recitedherein are inclusive of the numbers defining the range and include andare supportive of each integer within the defined range. The methods andtechniques described herein are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. See,e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990). All documents, or portions of documents, cited inthis application, including but not limited to patents, patentapplications, articles, books, and treatises, are hereby expresslyincorporated by reference. What is described in an embodiment of theinvention can be combined with other embodiments of the invention.

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

EXAMPLES Cell Culture

On day 0, CHO cells expressing a recombinant anti-TNFα antibody wereinoculated into 3 L bioreactors (Applikon, Foster City, CA) at 9.0 × 10⁶viable cells/mL in a working volume of 1500 ml of a serum-free,chemically-defined base medium. The cultures were maintained at 36° C.,DO at 30 mmHg, agitation at 400 RPM. The cell cultures were initiated inbatch mode and perfusion was started on day 3 using an ATF-2™alternating tangential flow filtration system (Refine Technologies,Hanover, NJ) equipped a 30 kDa NFWC GE RTP Hollow Fiber Cartridge (GEHealthcare, Pittsburg, PA). The medium was a serum-free,chemically-defined perfusion medium including manganese sulfatemonohydrate and cupric sulfate pentahydrate and pH as described inTable 1. The experiment was run in duplicate.

TABLE 1 pH Mn²⁺ (nM) Cu²⁺ (ppb) 6.85 50 10 6.85 50 100 6.85 1000 10 6.851000 100 7.0 50 10 7.0 50 100 7.0 1000 10 7.0 1000 100

The perfusion rate increased gradually from 0.3 to 1.0 workingvolume/day over the cell culture run. On day , the temperature wasshifted to 31° C. and the culture was harvested on day 17. Glucose wasmaintained between 4-8 g/L.

Samples were taken daily to assess the culture. The pH and partialpressure of CO₂ (pCO₂) and O₂ (pO₂) were measured using a Rapid Lab 1260blood gas analyzer (Siemens, Malvern, PA); concentration of glucose andlactate, were measured using a NovaFLEX (Nova Biomedical, Waltham, MA).Osmolality by determined by Model 2020 Osmometer (Advanced Instruments,Norwood, MA). Temperature, pH, dissolved oxygen and agitation werecontrolled using Applikon ADI1010 controllers.

On days 7, 10, 13, 15 and 17, 50 mL samples of the culture were removedfrom the bioreactors for product quality analysis. The samples werecentrifuged at 3000 rpm for 30 minutes at room temperature (BeckmanCoulter, Indianapolis, IN) and the supernatant was filtered through a0.2 µm tube top filter (Corning, Fisher Scientific, Pittsburgh, PA).Cell free supernatant was then frozen at -20° C. until thawed andProtein A purified prior to product quality analysis. Upon completion ofthe 17 day production, the remaining culture was removed from thebioreactors. Cells were separated from the supernatant by centrifugationat 3000 rpm for 30 minutes at 4° C. and the conditioned culture mediumwas sterile filtered using a 0.2 µm polyethersulfone (PES) cartridgefilter into Nalgene bottles (Fisher Scientific, Pittsburgh, PA), thenpurified by Protein and the neutralized eluates were tested as describedabove.

Viable cell density and cell viability were deterred by Vi-Cell (BeckmanCoulter, Brea, CA). Integrated viable cell density (IVCD) was calculatedas a cumulative viable cell density over the entire length of theproduction. Titer was measured using POROS® Protein A (LifeTechnologies, Grand Island, NY). Titer was determined in the supernatantand then adjusted for the volume that was occupied by the cells so thatit was representative of what was actually present in a given volume ofcell culture fluid. Since packed cell volume was expressed as a percentof the total volume, the PCV adjusted titers was always lower than thetiter in the supernatant.

Different N-glycan species were analyzed by hydrophilic-interactionliquid chromatography (HILIC) and are presented as a percent of thetotal peak area of the combined glycans. Antibody-containing sampleswere collected and purified by Protein A. The purified samples weretreated with PNGase-F and incubated at 37° C. for 2 hours to release theN-linked glycans. The enzymatically released glycans were labeled with2-aminobenzoic acid (2-AA) at 80° C. for 75 minutes. Excess 2-AA labelwas then removed with a Glycoclean S cartridge. The samples wereevaporated overnight and the resulting dry pellet was reconstituted withwater for subsequent HILIC analysis, using UPLC (Waters Corporation,Milford, MA). The glycans were injected and bound to the column in highorganic conditions and eluted with an increasing gradient of an aqueousammonium formate buffer. Fluorescence detection was used to monitor theglycan elution and the relative percentage of the major and minor glycanspecies were calculated. β-gal levels include A1G1F, A2G1F, A2G2F andthe analogous afucosylated forms. Afucosylated forms include A1G0, A2G0,A1G1, A2G1 and A2G2. Mannose 5 and Mannose 7 were also determined.

This experiment was designed to define the effects of each main factor(copper, manganese, and pH) and two way interactions. The experiment wasa three factor, two level (2³) full factorial design to define maineffects and two way interactions and did not include center points. Thestudy was intended to deliver power values of approximately 0.8 using asignal to noise ratio of 1.25. Profiles were generated using JMPstatistical software and Prediction Profiler (SAS Institute, Inc., Cary,NC).

Results

The concentration of copper and manganese in the perfusion medium didnot impact cell culture performance or productivity. While pH did notimpact cell growth or productivity, pH 6.85 reduced final viability byapproximately 10% (p<0.001), FIGS. 1-4 .

High Mannose Glycans

pH was the only factor that had a significant effect on high mannoselevels. As pH increased, so did the level of high mannose, see Table 2.

β-Galactosylation

The addition of manganese enhanced the β-galactosylation. The greaterthe concentration of manganese, the greater the percentage ofβ-galactosylation. pH had a statistically significant effect toβ-galactosylation as well. Increasing pH increased β-galactosylation butto a lesser extent than when compared to the increase when manganese wasadded, see FIG. 5 . The effect of copper on β-galactosylation wasinsignificant.

Afucosylation

Copper, manganese and pH all had a statistically significant impact onthe afucosylation levels. The greater the concentration of copper andmanganese and the higher the pH, the higher the level of afucosylation,see FIG. 6 .

All of the key glycans were significantly impacted by pH.β-galactosylation was significantly impacted by increasing manganeseconcentration. Increasing the level of manganese to its highest levelresulted in an increase of β-galactosylation by approximately 14% overthe base line, lowest level of copper and manganese tested at the samepH, as determined by statistical modeling. Afucosylation wassignificantly impacted by increasing both copper and manganeseconcentrations. Increasing the levels of copper and manganese enhancedthe level of afucosylation by approximately 1.3% over the base linevalue. While the addition of high concentrations of copper and manganesehad no impact on cell culture performance, they did have an impact onproduct quality. See Tables 2 and 3.

TABLE 2 Results from day 17 harvest Mn⁺² (nM) Cu⁺² (ppb) pHAfucosylation (%) High Mannose (%) β-galactosylation (%) 50 10 6.85 4.242.69 16.14 50 10 7.00 5.19 3.55 18.71 50 100 6.85 4.83 2.54 16.09 50 1007.00 5.93 3.32 21.90 1000 10 6.85 4.94 2.63 30.79 1000 10 7.00 6.26 3.2135.14 1000 100 6.85 5.46 2.53 29.31 1000 100 7.00 6.59 3.36 32.93

TABLE 3 Summary of the model fit (R²) and the statistical significanceof the terms that are part of the model (p values) Parameter Adjusted R²Higher pH Higher Mn²⁺ Higher Cu²⁺ P Values P Values P Valuesβ-Galactosylation 0.95 0.0028 <0.0001 -- Afucosylation 0.92 <0.0001<0.0001 0.0028 High Mannose 0.93 <0.0001 -- --

The designation of “higher” refers to a situation where pH, or otherfactors, are higher, then the different types of glycosylation go up.

Glycosylation can affect therapeutic efficacy of recombinant proteindrugs. It is well known that variations in Fc glycosylation can affectFc-mediated effector functions. Afucosylation and high mannose glycanscan enhance antibody-dependent cellular cytotoxicity (ADCC) activity.For use in a ADCC assay, afucosylated and fucosylated recombinantanti-TNFα antibody material was produced separately using a fed-batchprocess. Afucosylated antibody was made with the aid of an addedfucosyltransferase inhibitor. The resultant recombinant antibody wasabout 85% afucosylated. The afucosylated antibody material was thenmixed with completely fucosylated antibody material to produce specificlevels of afucosylation in the final antibody mixture. The antibodymaterial was then used to measure the level of ADCC activity at variouslevels of afucosylation to determine the sensitivity of the ADCCresponse.

The ADCC activity of the antibody mixture was evaluated in a cell-basedassay using CHO M7 cells that stably expressed a TNFα converting enzyme(TACE)-resistant form of transmembrane TNFα as target cells. NK92-M1cells, stably transfected with human CD16 (FcyRIIIa-158V) were used aseffector cells. Briefly, target cells were opsonized with increasingconcentrations (0.143 ng/mL to 40 ng/mL) of antibody prior toco-incubation with the NK92-M1/CD16 effector cells. Upon ADCC-mediatedtarget cell lysis, the intracellular enzyme adenylate kinase wasreleased into the cell culture medium. The amount of adenylate kinasereleased was measured using the ToxiLight™ Bioassay Kit (Lonza,Allendale, NJ). SoftMax® Pro (Molecular Devices, Sunnyvale, CA) was toperform a 4-parameter data analysis and a constrained model curve fit tothe dose-response data. Test sample activity was determined by comparingthe test sample response to the response obtained for the referencestandard and was reported as percent relative cytotoxicity.

For use in a complement-dependent cytotoxicity (CDC) assay,β-galactosylated material that was obtained from a chromatographicallyenriched fraction of the recombinant anti-TNFα antibody. The enrichedantibody was used to prepare solutions with specific levels ofβ-galactosylation. The level of CDC activity at various levels ofβ-galactosylation was then measured to establish the sensitivity of theCDC response.

The degree of CDC activity elicited by the antibody was evaluated in afunctional cell based assay. CHO M7 cells were pre-incubated with 20 µMcalcein-AM (Sigma, St. Louis, MO). The calcein-AM entered the cells andwas cleaved by nonspecific esterases to become fluorescent and trappedwithin the intact cell membranes. The calcein-loaded target cells wereincubated with different dose concentrations of the antibody (1.563ng/mL to 200 ng/mL), followed by complement addition (2.5% finalconcentration) for a second incubation.

After the complement incubation, the supernatant was removed and thefluorescence was measured using a microplate reader (EnVision, PerkinElmer, Waltham, MA). The fluorescence intensity was directlyproportional to the amount of cellular lysis. SoftMax® Pro (MolecularDevices, Sunnyvale, CA) was used to perform a 4-parameter data analysisand a constrained model curve fit to the dose response data. Test sampleactivity was determined by comparing the test sample response to theresponse obtained for the reference standard and was reported as percentrelative cytotoxicity.

Increasing the level of afucosylation by as little as 2% had a practicalimpact on ADCC activity (FIG. 7 ). CDC activity was also clearlyimpacted by increasing the level of β-galactosylation, although theresponse was much less sensitive (FIG. 8 ).

ADCC and CDC effector functions can be critical factors for the clinicalactivity of therapeutic proteins and achieving desired target values forspecific glycans may be key to reaching desired clinical endpoints.Small changes in afucosylation can have a big impact on the ADCCactivity of a glycoprotein. By altering copper and manganese it ispossible to control the levels of glycans that are responsible for theseeffector functions and direct the product quality.

1. A method for increasing the fucosylated glycan content on arecombinant protein comprising inoculating a bioreactor with host cellsexpressing the recombinant protein, culturing the host cells in a serumfree, chemically defined cell culture medium; increasing at least one ofthe following: the level of copper, the level of manganese, or the pH,harvesting the recombinant protein produced by the host cell.
 2. Themethod of claim 1, further comprising an increase in the level ofβ-galactosylation on the recombinant protein.
 3. The method according toclaim 1, wherein the concentration of copper is increased to 100 ppb. 4.The method according to claim 1, wherein the concentration of manganeseis increased to 1000 nM.
 5. The method according to claim 1, wherein thefucosylated glycan content is increased to influence the effectorfunction of the recombinant protein.
 6. The method according to claim 1,further comprising a temperature shift.
 7. The method according to claim6, wherein the temperature shift is from 36° C. to 31° C.
 8. The methodaccording to claim 6, wherein the temperature shift occurs at thetransition between the growth phase and production phase.
 9. The methodaccording to claim 6, wherein the temperature shift occurs during theproduction phase.
 10. The method according claim 1 wherein the host cellexpressing the recombinant protein is cultured in a batch culture,fed-batch culture, perfusion culture, or combinations thereof.
 11. Themethod according to claim 10, wherein the culture is a perfusionculture.
 12. The method according to claim 11, wherein perfusioncomprises continuous perfusion.
 13. The method according to claim 11,wherein the rate of perfusion is constant.
 14. The method according toclaim 11, wherein the perfusion is performed at a rate of less than orequal to 1.0 working volumes per day.
 15. The method according to claim11, wherein the perfusion is accomplished by alternating tangentialflow.
 16. The method according to claim 1, wherein the bioreactor has acapacity of at least 500 L.
 17. The method according to claim 1 whereinthe bioreactor has a capacity of at least 500 L to 2000 L.
 18. Themethod according to claim 1 wherein the bioreactor has a capacity of atleast 1000 L to 2000 L.
 19. The method according to claim 1, wherein thebioreactor is inoculated with at least 0.5 × 10⁶ cells/mL.
 20. Themethod according to claim 1, wherein the serum-free chemically definedcell culture medium is a perfusion cell culture medium.
 21. The methodaccording to claim 1, wherein the host cells are mammalian cells. 22.The method according to claim 1, wherein the host cells are ChineseHamster Ovary (CHO) cells.
 23. The method according to claim 1, whereinthe recombinant protein is a glycoprotein.
 24. The method according toclaim 1, wherein the recombinant protein is selected from the groupconsisting of a human antibody, a humanized antibody, a chimericantibody, a recombinant fusion protein, or a cytokine. 25-28. (canceled)29. The method according to claim 1, wherein the pH is increased to 7.0.